Abstract Stenotrophomonas maltophilia (Sm), a gram negative, multi-drug resistant bacterium, is increasingly recognized as an important opportunistic and nosocomial pathogen. Sm infection commonly manifests as pneumonia and blood stream infections but can also cause CNS, eye, skin, soft tissue, and urinary tract infections and act as a risk factor for lung exacerbations in Cystic Fibrosis (CF) patients. Despite this, there is minimal understanding of Sm virulence. My lab has demonstrated that Sm causes lung epithelial cell detachment and death and has also shown that Sm encodes a type II protein secretion system which triggers apoptosis in lung epithelial cells. After completing work regarding another Sm secreted factor, a siderophore that I determined to be a novel catecholate, I began characterizing a type IV secretion system (T4SS) encoded in the genome of clinical isolate K279a. I demonstrated that a mutation in a component of the Sm T4SS core complex (virB10) results in enhanced lung epithelial cell detachment and death indicating that the Sm T4SS inhibits cell death. I showed that the Sm T4SS inhibits staurosporine induced cell death as well as caspase activation and thus determined the Sm T4SS has an anti-apoptotic effect on human epithelial cells. I was able to confirm this phenotype upon Sm infection of primary human bronchial/tracheal epithelial cells. I tested the effect of the Sm T4SS on a human macrophage cell line (U937) and explanted macrophages obtained from A/J mouse femurs and determined that the Sm T4SS elaborates a pro-apoptotic effect on macrophages. Both phenotypes necessitated bacterial contact with the host cell and were evident in four other Sm clinical isolates. I also determined that neither apoptosis phenotype was attributed to a difference in wildtype (WT) vs virB10 mutant Sm attachment to the host cells. Moreover, I showed by intranasally infecting A/J mice with WT and virB10 mutant Sm that the T4SS enhanced the growth of Sm in mouse lungs. I also determined that the Sm T4SS confers a growth advantage when co-cultured with E. coli and P. aeruginosa as measured by colony forming units (CFUs) after a 24 hour co-culture. This is a phenotype that was previously only attributed to the type VI secretion system until recently when it was attributed to the Xanthomonas and Bartonella T4SS. In performing bioinformatic analysis using multiple software programs, I developed a list of 18 putative Sm T4SS effectors. By performing a bacterial two-hybrid assay, I determined that the 18 proteins indeed interact with the Sm T4SS apparatus via its coupling protein VirD4. Taken together, these data indicate Sm possesses a unique T4SS with three different cell-dependent phenotypes. Thus, I propose to i) determine the Sm T4SS effectors that modulate host cell apoptosis (Aim 1) and ii) assess Sm T4SS dependent growth advantages against P. aeruginosa and other bacteria that co-habitate the CF lung (Aim 2). Aside from providing critical understanding of Sm, this work will have implications for the many undefined T4SS that exist in the genomes of other bacteria and aid in our assessment of T4SS as a target for new anti-microbials.